ES2889874T3 - Treatment of optic neuropathy and reduction of steroid-induced oxidative stress with stabilized polyunsaturated substances - Google Patents

Abstract

A polyunsaturated substance for use in a method of treating glaucoma, steroid-induced cataracts, steroid-induced myopathy, or Smith-Lemli-Opitz syndrome (SLOS), wherein the method comprises: identifying a patient having glaucoma, cataract-induced due to steroids, steroid-induced myopathy, or Smith-Lemli-Opitz syndrome (SLOS) and requiring treatment; repeatedly administering a polyunsaturated substance to the patient or the patient's tissue, wherein the polyunsaturated substance is isotopically modified at one or more positions; wherein after said administration, the polyunsaturated substance is incorporated into the patient's body or the patient's tissue; wherein the polyunsaturated substance is a fatty acid, a fatty acid ester or a salt thereof.

Description

DESCRIPTION

Treatment of optic neuropathy and reduction of steroid-induced oxidative stress with stabilized polyunsaturated substances

Background

Country

Isotopically modified polyunsaturated fatty acids ("PUFAs") and other PUFAs modified to stabilize polyunsaturated substances in a patient, particularly for patients with oxidation-related disorders such as glaucoma, a nonhereditary optic neuropathy, steroid-induced oxidative stress, or disorders related to steroid-induced oxidation.

Description of Related Art

The number of diseases associated with oxidative stress is numerous and diverse, but it is well established that oxidative stress is caused by disturbances of the normal redox state within cells. An imbalance between the routine production and detoxification of reactive oxygen species ("ROS") such as peroxides and free radicals can result in oxidative damage to cellular structures and machinery. Under normal conditions, a potentially important source of ROS in aerobic organisms is activated oxygen leakage from mitochondria during normal oxidative respiration. Furthermore, macrophages and enzymatic reactions are also known to contribute to the generation of ROS within cells. Because cells and their internal organelles are bound to the lipid membrane, ROS can easily come into contact with membrane components and cause lipid oxidation. Ultimately, such oxidative damage can be transmitted to other biomolecules within the cell, such as DNA and proteins, through direct and indirect contact with active oxygen, oxidized membrane components, or other oxidized cellular components. Thus, one can easily imagine how oxidative damage can spread throughout a cell given the mobility of internal components and the interconnectedness of cellular pathways.

Lipid-forming fatty acids are well known as one of the main components of living cells. As such, they participate in numerous metabolic pathways and play an important role in a variety of pathologies. Polyunsaturated fatty acids ("PUFAs") are an important subclass of fatty acids. An essential nutrient is a food component that, directly or via conversion, fulfills an essential biological function and that is not produced endogenously or in quantities large enough to cover needs. For homeothermic animals, the two strictly essential PUFAs are the linoleic acids (cis,cis-9,12-Octadecadienoic acid; (9Z,12Z)-9,12-octadecadienoic acid; "LA"; 18:2; n-6 ) and alpha-linolenic acid (cis,cis,cis-9,12,15-octadecatrienoic acid; (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid; "ALA"; 18:3; n-3) , formerly known as vitamin F (Cunnane SC. Progress in Lipid Research 2003; 42:544-568). LA, by further enzymatic desaturation and elongation, is converted to higher n-6 PUFAs, such as arachidonic acid (AA; 20:4; n-6); while ALA gives rise to a higher n-3 series, including, but not limited to, eicosapentaenoic acid (EPA; 20:5; n-3) and docosahexaenoic acid (DHA; 22:6; n-3) ( Goyens PL et al Am J Clin Nutr 2006;84:44-53). Due to the essential nature of certain PUFAs or PUFA precursors, there are many known examples of their deficiency and these are often related to medical conditions. In addition, many PUFA supplements are available over the counter with proven efficacy against certain ailments (see, eg, US Patent No. 7,271,315 and US Patent No. 7,381,558).

PUFAs endow mitochondrial membranes with the proper fluidity necessary for optimal performance of oxidative phosphorylation. PUFAs also play an important role in the initiation and propagation of oxidative stress. PUFAs react with ROS through a chain reaction that amplifies an original event (Sun M, Salomon RG, J. Am. Chem. Soc. 2004; 126:5699-5708). However, the non-enzymatic formation of high levels of lipid hydroperoxides is known to result in several deleterious changes. In fact, coenzyme Q10 has been linked to increased PUFA toxicity through PUFA peroxidation and the toxicity of the resulting products (Do TQ et al, PNAS USA 1996; 93:7534-7539). Said oxidized products negatively affect the fluidity and permeability of their membranes; lead to oxidation of membrane proteins; and can be converted to a large number of highly reactive carbonyl compounds. The latter include reactive species such as acrolein, malonic, dialdehyde glyoxal, methylglyoxal, etc. (Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008; 153:6-20). But the most prominent products of PUFA oxidation are alpha, beta-unsaturated aldehydes such as 4-hydroxynon-2-enal (4-HNE; formed from n-6 PUFAs as LA or AA), 4-hydroxyhex- 2-enal (4HHE; formed from n-3 PUFAs as ALA or DHA) and the corresponding ketoaldehydes (Esterfbauer H et al. Free Rad. Biol. Med. 1991; 11:81-128; Long EK, Picklo MJ Free Rad Biol Med 2010;49:1-8). These reactive carbonyls cross-link (bio)molecules via Michael addition or Schiff base formation pathways, and have been implicated in a large number of pathological processes (such as those presented above), age-related conditions, and oxidative stress, and ageing. Importantly, in some cases, PUFAs appear to be oxidized at specific sites because the methylene groups of the 1,4-diene systems (the bis-allylic position) are substantially less stable to ROS and to enzymes such as cyclogenases and lipoxygenases than the methylenes. allyls.

Oxidation-resistant PUFAs, PUFA mimetics, PUFA prodrugs and/or oxidation-resistant PUFA-containing fats, and PUFA mimetics have now been discovered that are useful for stabilizing polyunsaturated substances in patients, particularly in patients with disorders related to obesity. oxidation or subjected to undesirable oxidative stress. .

Compendium

The present invention is defined in the appended claims.

Some embodiments provide a method of treating glaucoma, a nonhereditary optic neuropathy, steroid-induced oxidative stress, or a steroid-induced oxidation-related disorder, comprising identifying a patient who has glaucoma, a nonhereditary optic neuropathy, a steroid-induced oxidative stress disorder. steroid-induced oxidative steroid-induced stress or oxidation-related disorders requiring treatment; repeatedly administering a polyunsaturated substance to the patient or the patient's tissue, wherein the polyunsaturated substance is chemically modified such that one or more bonds are stabilized against oxidation; wherein after said administration, the polyunsaturated substance or a polyunsaturated metabolite thereof comprising said one or more chemical modifications is incorporated into the patient's body or the patient's tissue.

In some embodiments, the patient is classified as having a nonhereditary optic neuropathy. In some embodiments, the polyunsaturated substance is repeatedly administered to the patient beginning before surgery or the administration of a second therapeutic agent.

the polyunsaturated substance is repeatedly administered to the patient beginning at the same time as the surgical intervention or the administration of a second therapeutic agent

the polyunsaturated substance is repeatedly administered to the patient beginning after surgery or the administration of a second therapeutic agent

In some embodiments, the patient is classified as having glaucoma. In some embodiments, the nonhereditary optic neuropathy is glaucoma. In some embodiments, the glaucoma is primary glaucoma, developmental glaucoma, secondary glaucoma, or absolute glaucoma. In some embodiments, the glaucoma is primary angle-closure glaucoma, primary open-angle glaucoma, pigmentary glaucoma, or exfoliation glaucoma. In some embodiments, the glaucoma is primary congenital glaucoma, infantile glaucoma, or hereditary glaucoma. In some embodiments, the glaucoma is inflammatory glaucoma, phacogenic glaucoma, glaucoma secondary to intraocular hemorrhage, traumatic glaucoma, neovascular glaucoma, drug-induced glaucoma, or glaucoma of various origins.

In some embodiments, the patient is classified as having steroid-induced oxidative stress. In some embodiments, the polyunsaturated substance is repeatedly administered to the patient beginning before administration of a steroid for treatment of a disease or symptom. In some embodiments, the polyunsaturated substance is repeatedly administered to the patient beginning simultaneously with the administration of a steroid for treatment of a disease or symptom. In some embodiments, the polyunsaturated substance is repeatedly administered to the patient beginning after administration of a steroid for treatment of a disease or symptom. In some embodiments, the patient is classified as having a steroid-induced oxidation-related disorder. In some embodiments, the polyunsaturated substance treats, reduces, or inhibits cataracts or cataract formation. In some embodiments, the steroid-induced oxidation disorder is a cataract.

In some embodiments, the patient is a mammal. In other embodiments, the patient is a human. In other embodiments, the patient is a male. In other embodiments, the patient is a female.

In some embodiments, the polyunsaturated substance is a fatty acid, fatty acid mimetic, or fatty acid prodrug. In some embodiments, the fatty acid, fatty acid mimetic, or fatty acid prodrug is stabilized at one or more bis-allylic positions. In some embodiments, the stabilization comprises at least one 13C atom or at least one deuterium atom in a bis-allylic position, wherein at least one 13C atom or the at least one deuterium atom is present at a significantly lower level. higher than the naturally occurring level of abundance of that isotope. In some embodiments, the fatty acid prodrug is an ester. In some embodiments, the ester is a triglyceride, diglyceride, or monoglyceride. In some embodiments, the polyunsaturated substance is a fatty acid prodrug ester and the ester is an alkyl ester. In other embodiments, the polyunsaturated substance is a fatty acid prodrug ester and the ester is an ethyl ester.

In some embodiments, the stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprises between about 10% and 50% of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient. In other embodiments, the stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprise between about 10% and 30% of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient. In other embodiments, the stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprise about 20% or more of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient. In some embodiments, the stabilized fatty acid, fatty acid mimetic or fatty acid prodrug is isotopically stabilized.

In some embodiments, a patient's cell or tissue maintains a sufficient concentration of the fatty acid prodrug, fatty acid mimetic, or fatty acid to prevent autoxidation of the naturally occurring polyunsaturated fatty acid, mimetic, or ester prodrug.

In some embodiments, the polyunsaturated substance is an omega-3 fatty acid, fatty acid mimetic, or fatty acid prodrug, or an omega-6 fatty acid, fatty acid mimetic, or fatty acid prodrug. In some embodiments, the polyunsaturated substance is selected from the group consisting of 11,11-D2-linolenic acid, 14,14-D2-linolenic acid, 11,11,14,14-D4-linolenic acid, 11,11-D4-linolenic acid, D2-linolenic acid, 14,14-D2-linolenic acid, 11,11,14,14-D4-linolenic acid, 11-D-linolenic acid, 14-D-linolenic acid, 11,14-D2-linolenic acid, 11 -D-linoleic acid, 14-D-linoleic acid and 11,14-D2-linoleic acid. In some embodiments, the polyunsaturated substance is selected from the group consisting of 9,10,12,13,15,16-D6-linolenic acid, 9,10,11,12,13,15,16-D7-linolenic acid, 9,10,11,11,12,13,15,16-D8-linolenic acid, 9,10,12,13,14,14,15,16-D8-linolenic acid, 9,10,12,13 acid ,14,15,16-D7-linolenic acid, 9,10,11,11,12,13,14,14,15,16-D10-linolenic acid, 9,10,11,12,13,14,15 acid ,16-D8-linolenic acid, 9,10,12,13-D4-linolenic acid, 9,10,11,12,13-D5-linolenic acid, 9,10,11,11,12,13-D6- linoleic acid, or alkyl ester prodrug thereof. In some embodiments, the polyunsaturated substance is 9,10,12,13-D4-linolenic acid or an alkyl ester prodrug thereof. In some embodiments, the polyunsaturated substance is further stabilized at a pro-bis-allylic position.

In other embodiments, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions. In some embodiments, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions, wherein the chemical modification comprises at least one 13C atom at one or more bis-allylic positions or at least one deuterium atom at one or more bis-allylic positions. or more bis-allylic positions, and wherein the at least one 13C atom or the at least one deuterium atom is present at a level significantly higher than the naturally occurring abundance level of said isotope. In some embodiments, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions, wherein the chemical modification comprises at least one or more deuterium atoms at one or more bis-allylic positions, and wherein the al least one or more deuterium atoms are present at a level significantly above the naturally occurring abundance level of that isotope. In other embodiments, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions, wherein the chemical modification comprises two deuterium atoms at one or more bis-allylic positions, and wherein the two deuterium atoms are present at a level significantly above the naturally occurring level of abundance of that isotope.

In some embodiments, the polyunsaturated substance is chemically modified such that formation of a radical at an allylic or bis-allylic position on the polyunsaturated substance results in the radical delocalizing through at least two carbon atoms, in the that at least one of the carbon atoms is 13C or 12C covalently bonded to deuterium.

In some embodiments, all of the carbon atoms in the polyunsaturated substance are not isotopically modified or all of the hydrogen atoms in the polyunsaturated substance are not isotopically substituted.

In some embodiments, an antioxidant is co-administered. Such antioxidants include, but are not limited to, coenzyme Q, idebenone, mitoquinone, mitoquinol, vitamin C, or vitamin E. In some embodiments, the antioxidant is coenzyme Q, idebenone, mitoquinone, or mitoquinol. In other embodiments, the antioxidant is a mitochondria-targeted antioxidant. In some embodiments, the antioxidant is a vitamin, vitamin mimetic, or vitamin prodrug. In other embodiments, the antioxidant is a vitamin E, vitamin E mimetic, vitamin E prodrug, vitamin C, vitamin C mimetic, and/or vitamin C prodrug.

Detailed description of the preferred embodiment

Definitions:

As used herein, abbreviations are defined as follows:

4-HHE or HHE 4-Hydroxyhex-2-enal

4-HNE or HNE 4-Hydroxinon-2-enal

AA Arachidonic acid

ALA Alpha-linolenic acid

D Deuterated

D1 Mono-deuterated

D2 Di-deuterated

D2-LA Di-deuterated linoleic acid

Tri-deuterated D3

Tetra-deuterated D4

D5 Penta-deuterated

D6 Hexa-deuterated

DHA Docosahexaenoic acid (22:6; n-3)

EPA Eicosapentaenoic acid (20:5; n-3)

H-PUFA Non-deuterated polyunsaturated fatty acid

LA linoleic acid

PUFA(s) Polyunsaturated fatty acid(s)

ROS Reactive Oxygen Species

SNOMED Systematized Nomenclature of Medicine

TDMS Toxicology Data Management System

WT Wild Type

YPD Medium containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which these embodiments pertain. The terminology used in the description herein is to describe particular embodiments only and is not intended as a limitation of the embodiments. As used in the specification and appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless expressly stated otherwise, should be construed as open rather than limiting. As examples of the foregoing, the term "including" should be read as meaning "including, without limitation", "including but not limited to" or the like; the term "comprising", as used herein, is synonymous with "including", "containing" or "characterized by" and is inclusive or open-ended and does not exclude additional elements or method steps not recited; the term "having" should be interpreted as "having at least"; the term "includes" shall be interpreted as "includes but is not limited to"; the term "example" is used to provide exemplary instances of the article under discussion, not an exhaustive or limiting list thereof; and the use of terms such as 'preferably', 'preferred', 'desired' or 'desirable' and words of similar meaning should not be understood to imply that certain characteristics are critical, essential or even important to the structure or function of the invention, but is merely intended to highlight alternative or additional features that may or may not be used in a particular embodiment. Furthermore, the term "comprising" is to be construed as being synonymous with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the steps listed, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Similarly, a group of elements linked with the conjunction 'and' should not be read as requiring each and every one of those elements to be present in the grouping, but rather should be read as 'and/or' unless noted. expressly the opposite. Similarly, a group of elements linked with the conjunction 'or' should not be interpreted as requiring mutual exclusivity between that group, but should be read as 'and/or' unless expressly stated otherwise.

It is understood that, in any compound described herein having one or more chiral centers, if an absolute stereochemistry is not expressly indicated, then each center may be independently of the R configuration or S configuration or a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, enantiomerically enriched, racemic mixture, diastereomerically pure, diastereomerically enriched, or a stereoisomeric mixture. Furthermore, it is understood that, in any compound described herein having one or more double bonds that generate geometric isomers that can be defined as E or Z, each double bond can independently be E or Z a mixture thereof.

Likewise, it is understood that, in any compound disclosed, all tautomeric forms are also intended to be included.

It should be understood that when the compounds described herein have unfilled valencies, then the valences must be filled with hydrogens.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain comprising a fully saturated hydrocarbon group (no double or triple bonds). The alkyl group may have from 1 to 25 carbon atoms (wherever it appears herein, a numerical range such as "1 to 25" refers to each whole number in the given range, e.g., "1 to 25 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 25 carbon atoms, although the present definition also covers the occurrence of the term "alkyl " where a numerical range is not designated). The alkyl group can also be medium sized alkyl having 1 to 15 carbon atoms. The alkyl group could also be lower alkyl having 1 to 6 carbon atoms. The alkyl group of the compounds may be designated "C4" or "C1-C4 alkyl" or similar designations. By way of example only, "C1-C4 alkyl" indicates that there are from one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n- butyl, iso-butyl, sec-butyl, and t-butyl Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary-butyl, pentyl, and hexyl. The alkyl group may be substituted or unsubstituted.

As used herein, the terms "bis-allylic" or "bis-allylic position" generally refer to the position of a polyunsaturated substance, e.g. eg a polyunsaturated fatty acid or mimetic thereof, corresponding to the methylene groups of a 1,4-diene system.

As used herein, the terms "pro-bis-allylic" or "pro-bis-allylic position" refer to the methylene group that converts to the bis-allylic position upon enzymatic desaturation.

As used herein, the term "PUFA" refers to a polyunsaturated fatty acid. Unless otherwise specified, the term "PUFA" also includes esters of fatty acids. Accordingly, the term "PUFA" includes esters of pharmaceutically acceptable polyunsaturated fatty acids.

As used herein, the term "oxidation-related disorder" refers to disorders or symptoms of disorders caused by an imbalance between the routine production and detoxification of reactive oxygen species ("ROS"), such as peroxides. and free radicals, resulting in oxidative damage to cellular structures.

As used herein, the term "oxidative stress" refers to an imbalance between the routine production and detoxification of reactive oxygen species ("ROS"), such as peroxides and free radicals, resulting in oxidative damage to the cell structures. Consequently, "oxidative stress" leads to "oxidation-related disorders".

As used herein, the term "simultaneously" means within a few hours.

As used herein, the term "autoxidation" refers to autocatalytic oxidation, e.g. eg autocatalytic lipid peroxidation.

Optic neuropathies:

Optic neuropathy refers to damage to the optic nerve due to any cause. Damage and death of these nerve cells or neurons leads to the characteristic features of optic neuropathy. The main symptom is vision loss, with colors appearing with subtle discoloration in the affected eye. On medical examination, the optic nerve head can be visualized with an ophthalmoscope. A clear disc is characteristic of long-standing optic neuropathy. In many cases, only one eye is affected, and patients may not notice color vision loss until the doctor asks them to cover the good eye. Optic neuropathy is often called optic atrophy, to describe the loss of some or most of the optic nerve fibers. In medicine, "atrophy" usually means "shrunken but able to regrow", so some argue that "optic atrophy" as a pathological term is somewhat misleading, and the term "optic neuropathy" should be used instead. In short, optic atrophy is the end result of any disease that damages nerve cells anywhere between the retinal ganglion cells and the lateral geniculate body (anterior visual system).

Several optic neuropathies have been identified, including both inherited and non-inherited variants. As used herein, nonhereditary optic neuropathies are traumatic optic neuropathy; infiltrative optic neuropathy; compressive optic neuropathy; nutritional optic neuropathy; toxic optic neuropathy; optic neuritis; ischemic optic neuropathy; anterior ischemic optic neuropathy (AION); non-arterial AION (NAION); radiation optic neuropathy (RON); posterior ischemic optic neuropathy; and glaucoma. Nonhereditary optic neuropathies include Leber's hereditary optic neuropathy (LHON); dominant optic atrophy; and Behr's syndrome.

ROS-mediated DNA damage is implicated in a wide variety of disease states. the evidence now indicates that oxidative stress plays an important role in the development of various optic neuropathies, especially glaucoma. The pathogenic role of ROS in glaucoma is supported by several experimental findings, summarized in Izzoti et al., Mutation Research/Reviews in Mutation Research (2006) 612(2), 105-114. Evidence also suggests that oxidative DNA damage is significantly increased in the ocular epithelium that regulates outflow of aqueous humor from glaucoma patients. It is now recognized that increased oxygen levels, light exposure and high PUFA content lead to increased PUFA peroxidation in ocular tissues. Certain classes of polyunsaturated substances have previously been discovered to be particularly suitable for treating, reducing or inhibiting the effects of oxidative stress and oxidation-related disorders. See, for example, WO 2007/102030; WO 2011/053870; WO 2012/148926; WO 2012/148930; WO 2012/148927; and WO 2012/148929. Accordingly, some aspects of the present embodiments arise from the discovery that stabilized substances, such as stabilized PUFAs, are useful for treating glaucoma or non-inherited optic neuropathies. These stabilized substances are particularly useful for treating glaucoma. In some cases, glaucoma is hereditary. Said stabilized substances are especially useful for administering to patients before, during or after the development of a non-hereditary optic neuropathy. Furthermore, said substances are particularly useful for treating diseases and secondary effects promoted, facilitated or caused by conventional treatments of non-hereditary optic neuropathies.

Steroid-induced oxidative stress and oxidation-related disorders:

Steroids are taken or administered for a variety of therapeutic uses, including the treatment, suppression, inhibition, or use in allergic diseases; as antiemetics; as antineoplastics; in autoimmune disorders, such as destruction of erythrocytes; in bronchial asthma; in cerebral edema; in congenital adrenal hyperplasia; in gastrointestinal diseases; in liver diseases; in eye diseases; in organ transplantation; in kidney diseases; in substitution therapies; in inflammation; in rheumatic disorders; in sarcoidosis; in skin diseases; in cerebrovascular accidents and spinal cord injuries; and in thrombocytosis.

However, steroids also cause cataract formation; glaucoma; and myopathy; among other toxicities and side effects. See Islam et al. Eye (2007) 21, 321-323; Dickerson, Jr. et al. Exp. Eye Res. (1997) 65, 507-516; Haller et al. Arch Opththalmol (2010) 128(3). Indeed, cataracts are a well-established complication of glucocorticoid therapy, related to both dose and duration of therapy. See Bucala et al. Exp. Eye Res. (1985) 40, 853-863; James, J. Ocular Pharm. & Ther. (2007) 23, 403-420. Children are at particular risk. Additionally, cessation of steroid therapy may not reverse developed opacities, and cataracts may progress despite reduction or cessation of therapy. Standard treatment protocols recommend that patients receiving glucocorticoid therapy undergo periodic slit-lamp examinations for steroid-induced cataracts, especially posterior subcapsular cataracts. Studies indicate that multifunctional antioxidants are capable of delaying cataract formation in animal models. See Randazzo et al. "Orally Active Multi-Functional Antioxidants Delay Cataract Formation in Streptoxotocin (Type 1) Diabetic and Gamma-Irradiated Rats," PLoS One (April 2011) 6(4), e18980. However, as discussed below, the stochastic nature of antioxidants limits their applicability to treating disorders caused by oxidative stress.

In addition, myopathy, characterized by weakness of proximal limb muscles, is occasionally seen in patients taking large doses of steroids, especially corticosteroids, and is also clinically relevant for patients with endogenous Cushing's syndrome. Such myopathy may be severe enough to impair ambulation and is used as an indication for discontinuation of therapy. In addition, steroid myopathy is also a cause for concern in patients suffering from asthma or chronic obstructive pulmonary disease, with said complication decreasing respiratory function. Recovery from steroid myopathies can be slow and ultimately incomplete.

As mentioned above, steroids, and especially glucocorticoids and corticosteroids, have an important role in the management of inflammatory eye diseases. In fact, after glaucoma filtering surgery, topical steroids are often used to delay the wound healing process by decreasing fibroblast infiltration, thus reducing potential scarring. However, steroids are also believed to induce glaucoma, but the pathophysiology of steroid-induced glaucoma is not particularly well understood. Consequently, a therapeutic regimen that allows the use of steroids after glaucoma filtering surgery, but does not increase the likelihood of glaucoma, would be desirable. Similarly, steroid therapy or hormone therapy also plays an important role in the treatment and regulation of fertility cycles and fertility disorders. Oxidative stress, however, is a particularly troublesome symptom of infertility. See Lombardo et al. Asian Journal of Andrology (2011) 13, 690-697; See Storey, Int. J. Biol. (2008), 52(5-6), 427-437; Ruder et al., Curr Opin Obstet Gynecol (2009) 21(3), 219-222. Therefore, a therapeutic regimen that allows the use of steroids, but does not increase the likelihood of infertility, would also be desirable.

Some steroids are known to react with ROS and are especially susceptible to peroxidation reactions. See Liu et al. J. Lipid Res. (2013) 54, 244-253. As steroids are important components of lipid membranes, the susceptibility of steroids to oxidative stress leads to increased cell damage of important membrane components such as fatty acids. Previously, certain classes of polyunsaturated substances have been discovered that are particularly suitable for treating, reducing or inhibiting the effects of oxidative stress. and oxidation-related disorders. See, p. eg, WO 2007/102030; WO 2011/053870; WO 2012/148926; WO 2012/148930; WO 2012/148927; and WO 2012/148929. Accordingly, aspects of the present embodiments arise from the discovery that stabilized substances, such as stabilized PUFAs, are useful for treating oxidative stress and oxidation-related disorders resulting from steroid oxidation. Such stabilized substances are especially useful for administering to patients before, during, or after steroid administration. Furthermore, such substances are particularly useful for treating diseases and side effects promoted, facilitated or caused by the administration of steroids.

Smith-Lemli-Opitz syndrome (SLOS) is an example of a steroid-induced oxidation-related disorder caused by mutations in the gene encoding 3p-hydroxysterol-A7-reductase, and as a result of this defect, the 7 -dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. See Liu et al., J. Lipid Res. (2013) 54, 244-253. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cellular and animal models of SLOS. Id. Accordingly, some aspects of the present embodiments arise from the discovery that stabilized substances, such as stabilized PUFAs, are useful for treating conditions associated with SLOS, such as increased levels of oxidative stress. Such stabilized substances are especially useful for administering to patients before, during or after the administration of conventional therapy.

Compositions and methods:

Some aspects of the embodiments arise from: (1) the realization that while polyunsaturated substances such as essential PUFAs are vital for the proper function of lipid membranes, and in particular mitochondrial membranes, their inherent drawback, i.e. , the propensity to be oxidized by ROS with detrimental result, is implicated in a variety of oxidation-related disorders such as glaucoma, nonhereditary optic neuropathies, steroid-induced oxidative stress, and steroid-induced oxidation-related disorders; (2) antioxidants cannot prevent PUFA peroxidation due to the stochastic nature of the process and the stability of PUFA peroxidation products (reactive carbonyls) to antioxidant treatment, and (3) ROS-induced damage of prone sites. to oxidation within PUFAs can be overcome by using an approach that makes them less susceptible to such oxidations, without compromising any of their beneficial physical properties. Indeed, some aspects of the embodiments arise from the understanding that formation of a radical at an allylic or bis-allylic position of a polyunsaturated substance results in the radical delocalizing through at least two carbon atoms. Consequently and unexpectedly, embodiments possessing stabilized bonds and/or atoms within the area of radical delocalization exhibit improved stability to oxidative stress. Some aspects of the embodiments describe the use of the isotopic effect to achieve stabilizing effects, only at the essential PUFA and PUFA precursor sites that are most important for oxidation, while other aspects contemplate sites other than those that matter most for oxidation. oxidation.

One embodiment provides a method of treating glaucoma, a non-hereditary optic neuropathy, steroid-induced oxidative stress, or a steroid-induced oxidation-related disorder, comprising identifying a patient who has glaucoma, a non-hereditary optic neuropathy, a steroid-induced oxidative stress. steroid-induced oxidative or steroid-induced oxidation-related disorders in need of treatment; repeatedly administering a polyunsaturated substance to the patient or the patient's tissue, wherein the polyunsaturated substance is chemically modified such that one or more bonds are stabilized against oxidation; wherein after said administration, the polyunsaturated substance or a polyunsaturated metabolite thereof comprising said one or more chemical modifications is incorporated into the patient's body or the patient's tissue.

In another embodiment, the patient is classified as having a nonhereditary optic neuropathy. In one embodiment, the polyunsaturated substance is repeatedly administered to the patient beginning before surgery or the administration of a second therapeutic agent.

polyunsaturated is repeatedly administered to the patient beginning simultaneously with the surgical intervention or the administration of a second therapeutic agent.

polyunsaturated is repeatedly administered to the patient beginning after surgery or the administration of a second therapeutic agent.

In another embodiment, the patient is classified as having glaucoma. In some embodiments, the nonhereditary optic neuropathy is glaucoma. In some embodiments, the glaucoma is primary glaucoma, developmental glaucoma, secondary glaucoma, or absolute glaucoma. In some embodiments, the glaucoma is primary angle-closure glaucoma, primary open-angle glaucoma, pigmentary glaucoma, or exfoliation glaucoma. In some embodiments, the glaucoma is primary congenital glaucoma, infantile glaucoma, or hereditary glaucoma. In some embodiments, the glaucoma is inflammatory glaucoma, phacogenic glaucoma, glaucoma secondary to intraocular hemorrhage, traumatic glaucoma, neovascular glaucoma, drug-induced glaucoma, or glaucoma of various origins.

By way of description, primary glaucoma includes primary angle closure glaucoma, also known as primary angle closure glaucoma, narrow angle glaucoma, pupillary block glaucoma, acute congestive glaucoma. Examples are acute angle-closure glaucoma; chronic angle-closure glaucoma; intermittent angle-closure glaucoma; superimposed on chronic open-angle closure glaucoma. Primary glaucoma also includes primary open angle glaucoma, also known as chronic open angle glaucoma, simple chronic glaucoma, simple glaucoma. Examples are high tension glaucoma and low tension glaucoma. Primary glaucoma also includes variants of pigmentary glaucoma and exfoliation glaucoma, also known as pseudoexfoliative glaucoma or capsular glaucoma. Developmental glaucoma includes primary congenital glaucoma, infantile glaucoma, and glaucoma associated with hereditary family diseases ("hereditary glaucoma"). Secondary glaucoma includes inflammatory glaucoma, such as uveitis of all types and Fuchs' heterochromic iridocyclitis; phacogenic glaucoma, such as angle-closure glaucoma with mature cataract, phacoanaphylactic glaucoma secondary to lens capsule rupture, phacolytic glaucoma due to blockage of the phacotoxic network, and lens subluxation; glaucoma secondary to intraocular hemorrhage, such as hyphema and hemolytic glaucoma, also known as erythroclassical glaucoma; traumatic glaucoma, such as angular recession glaucoma, postsurgical glaucoma, aphakic pupillary block, and ciliary block glaucoma; neovascular glaucoma; drug-induced glaucoma, such as corticosteroid-induced glaucoma and alpha-chymotrypsin glaucoma; and glaucoma of various origins, such as that associated with intraocular tumors, retinal detachments, glaucoma secondary to severe chemical burns of the eye, that associated with essential atrophy of the iris, and toxic glaucoma. Absolute glaucoma is the final stage of all types of glaucoma, characterized by the eye having no vision, absent pupillary light reflex and pupillary response, and having a stony appearance.

In another embodiment, the patient is classified as having steroid-induced oxidative stress. In one embodiment, the polyunsaturated substance is repeatedly administered to the patient beginning before administration of a steroid for treatment of a disease or symptom. In one embodiment, the polyunsaturated substance is repeatedly administered to the patient beginning simultaneously with the administration of a steroid for the treatment of a disease or symptom. In one embodiment, the polyunsaturated substance is repeatedly administered to the patient beginning after the administration of a steroid for the treatment of a disease or symptom. In one embodiment, the patient is classified or further classified as having a steroid-induced oxidation-related disorder.

In another embodiment, the polyunsaturated substance is a fatty acid, fatty acid mimetic, or fatty acid prodrug.

In another embodiment, the fatty acid, fatty acid mimetic, or fatty acid prodrug is stabilized at one or more bis-allylic positions.

In another embodiment, the stabilization comprises at least one 13C atom or at least one deuterium atom in a bis-allylic position, wherein the at least one 13C atom or the at least one deuterium atom is present at a level significantly higher than the naturally occurring level of abundance of that isotope.

In another embodiment, the stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprises between about 10% and 50% of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient.

In another embodiment, the isotopically stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprises between about 10% and 30% of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient.

In another embodiment, the isotopically stabilized fatty acid, fatty acid mimetic, or fatty acid prodrug comprises about 20% or more of the total amount of fatty acid, fatty acid mimetic, or fatty acid prodrug administered to the patient.

In another embodiment, a patient cell or tissue maintains a sufficient concentration of the fatty acid, fatty acid mimetic, or fatty acid prodrug to prevent autoxidation of the naturally occurring polyunsaturated fatty acid, ester mimetic, or prodrug.

In another embodiment, the polyunsaturated substance is an omega-3 fatty acid, fatty acid mimetic or fatty acid prodrug, or an omega-6 fatty acid, fatty acid mimetic or fatty acid prodrug.

In another embodiment, the polyunsaturated substance is selected from the group consisting of 11,11-D2-linolenic acid, 14,14-D2-linolenic acid, 11,11,14,14-D4-linolenic acid, 11,11-D4-linolenic acid, D2-linolenic acid, 14,14-D2-linolenic acid, 11,11,14,14-D4-linolenic acid, 11-D-linolenic acid, 14-D-linolenic acid, 11,14-D2-linolenic acid, 11 -D-linoleic acid, 14-D-linoleic acid and 11,14-D2-linoleic acid.

In another embodiment, the polyunsaturated substance is further stabilized at a pro-bis-allylic position.

In another embodiment, the fatty acid prodrug is an ester.

In another embodiment, the ester is a triglyceride, diglyceride, or monoglyceride.

In another embodiment, an antioxidant is co-administered.

In another embodiment, the co-administered antioxidant is coenzyme Q, idebenone, mitoquinone, mitoquinol, vitamin C, or vitamin E.

In another embodiment, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions.

In another embodiment, the polyunsaturated substance is selected from the group consisting of 9,10,12,13,15,16-D6-linolenic acid, 9,10,11,12,13,15,16-D7-linolenic acid, 9,10,11,11,12,13,15,16-D8-linolenic acid, 9,10,12,13,14,14,15,16-D8-linolenic acid, 9,10,12,13 acid ,14,15,16-D7-linolenic acid, 9,10,11,11,12,13,14,14,15,16-D10-linolenic acid, 9,10,11,12,13,14,15 acid ,16-D8-linolenic acid, 9,10,12,13-D4-linolenic acid, 9,10,11,12,13-D5-linolenic acid, 9,10,11,11,12,13-D6- linoleic acid, or alkyl ester prodrug thereof.

In another embodiment, the polyunsaturated substance is 9,10,12,13-D4-linolenic acid or an alkyl ester prodrug thereof.

In another embodiment, the polyunsaturated substance is further chemically modified at one or more bisallylic positions, wherein the chemical modification comprises at least one 13C atom at one or more bisallylic positions or at least one deuterium atom at one or more bisallylic positions. more bis-allylic positions, and wherein the at least one 13C atom or the at least one deuterium atom is present at a level significantly higher than the naturally occurring abundance level of said isotope.

In another embodiment, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions, wherein the chemical modification comprises at least one or more deuterium atoms at the one or more bis-allylic positions, and wherein the at least one or more deuterium atoms are present at a level significantly above the naturally occurring abundance level of said isotope.

In another embodiment, the polyunsaturated substance is further chemically modified at one or more bis-allylic positions, and wherein the chemical modification comprises two deuterium atoms at one or more bisallylic positions, and wherein both deuterium atoms are present. at a level significantly above the naturally occurring level of abundance of that isotope.

In another embodiment, all carbon atoms of the polyunsaturated substance are not isotopically modified. In other embodiments, all of the hydrogen atoms in the polyunsaturated substance are not isotopically substituted. In another embodiment, at least one carbon atom is not 13C. In another embodiment, at least one hydrogen atom is not deuterium.

In another embodiment, the polyunsaturated substance is 9,10,12,13-D4-linolenic acid or an alkyl ester prodrug thereof.

In some embodiments, an isotopically modified polyunsaturated fatty acid or mimetic refers to a compound having structural similarity to a naturally occurring PUFA that is stabilized chemically or by reinforcement with one or more isotopes, eg 13C and/or or deuterium. Generally, if deuterium is used as a booster, one or both hydrogens on a methylene group can be boosted.

In some embodiments, the patient is a mammal (ie, a human, domesticated mammal, horse, dog, cow, etc.). In some embodiments, the mammal is a male. In other embodiments, the mammal is a female. In some embodiments, sperm survival or sperm motility is increased.

In some embodiments, the probability of fertilization of an egg by sperm is increased. In some embodiments, the probability increases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100% or a range limited by any two of the numbers listed above, or approximately any of the numbers or ranges listed above. In some embodiments, the probability is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, or approximately any of the numbers or ranges listed above.

Some aspects of this invention provide compounds that are essential PUFA analogs with one, several, or all of the bis-allylic positions substituted with heavy isotopes. In some embodiments, the CH2 groups, which will be converted at the bis-allylic position in a PUFA upon enzymatic conversion, are replaced with one or two heavy isotopes.

In some embodiments, the chemical identity of the PUFAs, that is, the chemical structure without regard to isotope substitutions or substitutions that mimic isotope substitutions, remains the same upon ingestion. For example, the chemical identity of essential PUFAs, ie PUFAs that are not generally synthesized by mammals such as humans, may remain identical after ingestion. In some cases, however, PUFAs can be further extended/desaturated in mammals, thus changing their chemical identity upon ingestion. Similarly with mimetics, the chemical identity may remain unchanged or may be subject to similar extension/desaturation. In some embodiments, PUFAs that extend, and optionally desaturate, after ingestion and further metabolism they may be referred to as higher homologues.

In some embodiments, the level of naturally occurring abundance refers to the level of isotopes, eg, 13C and/or deuterium, that can be incorporated into polyunsaturated substances, such as PUFAs, which would be relative to the natural abundance of the isotope in nature. . For example, 13C has a natural abundance of about 1% 13C atoms on total carbon atoms. Therefore, the relative percentage of carbon that has a greater than natural abundance of 13C in polyunsaturated substances may have a greater than natural abundance level of about 1% of their total 13C-reinforced carbon atoms, such as 2 %, but preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, %, or a range limited by two of the aforementioned numbers, of 13C with respect to one or more carbon atoms in each molecule of polyunsaturated substance. In other embodiments, the percentage of total 13C-reinforced carbon atoms is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

With respect to hydrogen, in some embodiments, deuterium has a natural abundance of about 0.0156% of all naturally occurring hydrogen in Earth's oceans. Therefore, a polyunsaturated substance, such as a PUFA, that has a greater than natural abundance of deuterium can be greater than this level or greater than the natural abundance level of about 0.0156% of its deuterium-reinforced hydrogen atoms, such as 0.02%, but preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, %, or a range limited by any two of the numbers listed above, of deuterium with respect to one or more hydrogen atoms in each molecule polyunsaturated substance. In other embodiments, the percentage of total deuterium-reinforced hydrogen atoms is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100.

In some aspects, a composition of polyunsaturated substances, such as PUFAs, contains both isotopically modified polyunsaturated substances and isotopically unmodified polyunsaturated substances. Isotopic purity is a comparison between a) the relative number of molecules of isotopically modified polyunsaturated substances and b) the total molecules of both isotopically modified polyunsaturated substances and polyunsaturated substances without heavy atoms. In some embodiments, isotopic purity refers to polyunsaturated substances that are otherwise the same except for heavy atoms.

In some embodiments, isotopic purity refers to the percentage of molecules of isotopically modified polyunsaturated substances, such as PUFAs, in the composition relative to the total number of molecules of the isotopically modified polyunsaturated substances plus polyunsaturated substances without heavy atoms. For example, the isotopic purity can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95%, or 100%, or a range limited by any two of the numbers listed above. In other embodiments, the isotopic purity is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the isotopic purity of the polyunsaturated substances can be about 10%-100%, 10%-95%, 10%-90%, 10%-85%, 10%-80%, 10%-75%. , 10%-70%, 10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, 10%-40%, 10%-35%, 10 %-30%, 10%-25% or 10%-20% of the total number of molecules of the polyunsaturated substances in the composition. In other embodiments, the isotopic purity of the polyunsaturated substances can be about 15%-100%, 15%-95%, 15%-90%, 15%-85%, 15%-80%, 15%-75%. , 15%-70%, 15%-65%, 15%-60%, 15%-55%, 15%-50%, 15%-45%, 15%-40%, 15%-35%, 15 %-30%, 15%-25% or 15%-20% of the total number of molecules of the polyunsaturated substances in the composition. In some embodiments, the isotopic purity of the polyunsaturated substances can be about 20%-100%, 20%-95%, 20%-90%, 20%-85%, 20%-80%, 20%-75%. , 20%-70%, 20%-65%, 20%-60%, 20%-55%, 20%-50%, 20%-45%, 20%-40%, 20%-35%, 20 %-30%, or 20%-25% of the total number of molecules of the polyunsaturated substances in the composition. Two molecules of an isotopically modified polyunsaturated substance out of a total of 100 total molecules of isotopically modified polyunsaturated substances plus polyunsaturated substances without heavy atoms will have 2% isotopic purity, regardless of the number of heavy atoms contained in the two isotopically modified molecules.

In some aspects, an isotopically modified polyunsaturated substance, such as a PUFA molecule, may contain a deuterium atom, such as when one of the two hydrogens on a methylene group is replaced by deuterium, and may therefore be referred to as a "D1" PUFA. ". Similarly, an isotopically modified PUFA molecule may contain two deuterium atoms, such as when the two hydrogens of a methylene group are replaced by deuterium, and thus may be referred to as a "D2" PUFA. Similarly, an isotopically modified PUFA molecule may contain three deuterium atoms and may be referred to as a "D3" PUFA. Similarly, an isotopically modified PUFA molecule may contain four deuterium atoms and may be referred to as a "D4" PUFA. In some embodiments, an isotopically modified PUFA molecule may contain five deuterium atoms or six deuterium atoms and may be referred to as a "D5" or "D6" PUFA, respectively.

The number of heavy atoms in a molecule, or the isotopic charge, can vary. For example, a molecule with a relatively low isotopic charge may contain about 1, 2, 3, 4, 5, or 6 deuterium atoms. A molecule with moderate isotopic charge may contain approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 deuterium atoms. In a highly charged molecule, each hydrogen can be replaced with a deuterium. Therefore, isotopic charge refers to the percentage of heavy atoms in each polyunsaturated substance, such as a PUFA molecule. For example, the isotopic charge can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95%, or 100% of the number of same type of atoms compared to a PUFA without heavy atoms of the same type (e.g., hydrogen would be "same type "than deuterium). In some embodiments, the isotopic loading is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95% or 100%. Unintended side effects are expected to be reduced when there is high isotopic purity in a PUFA composition but low isotopic charge in a given molecule. For example, metabolic pathways are likely to be less affected by the use of a PUFA composition with high isotopic purity but low isotopic loading.

It will be readily appreciated that when one of the two hydrogens of a methylene group is replaced with a deuterium atom, the resulting compound may possess a stereocenter. In some embodiments, it may be desirable to use racemic compounds. In other embodiments, it may be desirable to use enantiomerically pure compounds. In additional embodiments, it may be desirable to use diastereomerically pure compounds. In some embodiments, it may be desirable to use mixtures of compounds having enantiomeric excesses and/or diastereomeric excesses of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. %, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range limited by any two of the above numbers. In other embodiments, enantiomeric excesses and/or diastereomeric excesses are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or a range limited by any two of the above numbers. In some embodiments, it may be preferable to use stereochemically pure enantiomers and/or diastereomers of the embodiments, such as when contact with chiral molecules is directed at attenuating oxidative damage. However, in many circumstances, non-chiral molecules are being targeted to attenuate oxidative damage. Under such circumstances, the embodiments can be used without concern for their stereochemical purity. Furthermore, in some embodiments, mixtures of enantiomers and diastereomers can be used even when the compounds are chiral molecules targeted at attenuating oxidative damage.

In some aspects, isotopically modified polyunsaturated substances impart a number of heavy atoms in a particular fabric. Thus, in some respects, the amount of heavy molecules will be a particular percentage of the same type of molecules in a tissue. For example, the number of heavy molecules may be about 1%-100% of the total amount of the same type of molecules. In some aspects, 10-50% of the molecules are replaced by the same type of heavy molecules.

In some embodiments, a compound with the same chemical bond structure as an essential PUFA but with a different isotopic composition at particular positions will have significantly and usefully different chemical properties from the unsubstituted compound. Particular positions with respect to oxidation, including oxidation by ROS, comprise bis-allylic positions of essential polyunsaturated fatty acids and their derivatives, as shown below as the indicated compound (1). Essential PUFAs reinforced isotopically at the bis-allylic positions shown below will be more stable to oxidation.

Some embodiments provide particular methods of using compounds indicated as (2) or salts thereof, while the sites may be further reinforced with carbon-13. R1 = alkyl, H or cation; m = 1-10; n = 1-5, where at each bis-allylic position, one or both Y atoms are deuterium atoms, e.g.

11,11-dideutero-cis,cis-9,12-octadecadienoic acid (11,11-dideutero-(9Z,12Z)-9,12-octadecadienoic acid; D2-LA); and 11,11,14,14-tetradeutero-cis,cis,cis-9,12,15-octadecatrienoic acid (11,11,14,14-tetradeutero-(9Z,12Z,15Z)-9,12,15- -octadecatrienoic; D4-ALA).

In some embodiments, such positions, in addition to deuteration, may be further strengthened by carbon-13, each at isotope abundance levels above the naturally occurring abundance level. All other carbon-hydrogen bonds in the PUFA molecule may optionally contain deuterium and/or carbon-13 at or above the level of natural abundance.

Essential PUFAs are biochemically converted to higher homologues by desaturation and elongation. Therefore, some non-bis-allylic sites in the precursor PUFAs will become bis-allylic upon biochemical transformation. These sites then become sensitive to oxidation, including oxidation by ROS. In a further embodiment, said pro-bis-allylic sites, in addition to existing bis-allylic sites, are strengthened by isotope substitution as shown below. Consequently, this aspect of the invention provides the use of compounds indicated as (3) or salts thereof, where in each bis-allylic position, and in each pro-bis-allylic position, one or more of the X atoms o Y can be deuterium atoms. R1 = alkyl, cation or H; m = 1-10; n = 1-5; p = 1-10.

R = H, C3H7; R1 = H, alquilo o catión; Y = H o D; X = H o D

R=H, C3H7; R1 = H, alkyl or cation; Y = H or D; X = H or D

Such positions, in addition to deuteration, can be further strengthened with carbon-13, each at isotope abundance levels above the naturally occurring abundance level. In some embodiments, one or more other carbon-hydrogen bonds in the PUFA molecule may independently contain deuterium and/or carbon-13 at or above the level of natural abundance.

PUFA oxidation at different bis-allylic sites gives rise to different sets of oxidation products. For example, 4-HNE is formed from n-6 PUFA, while 4-HHE is formed from n-3 PUFA (Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008; 153:6 -twenty). The products of said oxidation have different regulatory, toxic, signaling, etc., properties. Therefore, it is desirable to control the relative extent of such oxidations. Consequently, some aspects of the invention provide for the use of compounds indicated as (6) or salts thereof, differentially reinforced with heavy stable isotopes at selected bis-allylic or pro-bis-allylic positions, to control the relative yield of oxidation at different sites, as shown below, such that any of the Y1-Yn and/or X1-Xm pairs at the bis-allylic or pro-bis-allylic positions of the PUFAs may contain deuterium atoms. R1 = alkyl, cation or H; m = 1-10; n = 1-6; p = 1-10

⁽6 ⁾

⁽ 6 ⁾

R=H, C3H7; R1 = H, alkyl or cation; Y = H or D; X = HOD

Such positions, in addition to deuteration, can be further strengthened with carbon-13. In some embodiments, one or more other carbon-hydrogen bonds in the PUFA molecule may independently contain deuterium and/or carbon-13 at or above the level of natural abundance. It will be appreciated that the breaklines in the structure shown above represent a PUFA with a varying number of double bonds, a varying number of total carbons, and a varying combination of isotope-reinforced bis-allylic and pro-bis-allylic sites.

The exact structures of the compounds illustrated above are shown below providing isotope-reinforced n-3 (omega-3) and n-6 (omega-6) essential polyunsaturated fatty acids, and the PUFAs made from them biochemically by desaturation. /elongation. Any of these compounds can be used to slow, inhibit, reduce, or prevent oxidation. In the following compounds, PUFAs are isotopically reinforced at oxidation-sensitive sites and/or sites that may become oxidation-sensitive upon biochemical desaturation/elongation. R1 can be H, alkyl or cation; R2 can be H or D; * represents 12C or 13C, in which at least one deuterium or at least one 13C is present above the naturally occurring level of abundance.

D-linoleic acids include:

Subsequent per-deuterated linoleic acid can be produced by microbiological methods, for example, by culture in media containing deuterium and/or carbon-13.

D-arachidonic acids include:

Subsequent per-deuterated arachidonic acid can be produced by microbiological methods, such as culture in media containing deuterium and/or carbon-13.

Post-per-deuterated linolenic acid can be produced by microbiological methods, such as culture in media containing deuterium and/or carbon-13.

In some aspects of the invention, any PUFA, essential or not, that is capable of being absorbed from the diet and utilized in the body can be used. In the case of essential or non-essential PUFAs or precursors, supplemented stabilized materials may compete with other dietary absorption and bio-manufacturing to reduce available concentrations of disease-causing species.

In some aspects of the invention, polyunsaturated compounds are reinforced at oxidation-sensitive positions, as described by the structures above, are heavy isotopes enriched at such positions compared to the natural abundance of the appropriate isotope, deuterium and/or carbon-13.

In some embodiments, the disclosed compounds are enriched to 99% isotope purity or greater. In some embodiments, the heavy isotope enrichment at said positions is between 50%-99% deuterium and/or carbon-13.

In some embodiments, modified fatty acids, when dosed through the diet as drugs or supplements, may be dosed as prodrugs, including, but not limited to, pharmaceutically suitable, non-toxic esters of the parent fatty acid or mimetic, such as a ethyl ester or glyceryl ester. East Ester aids drug tolerance in the intestine, aids digestion, and relies on high levels of esterases in the intestines to de-esterify ester prodrugs into the active acid form of the drug being adsorbed. Thus, in some embodiments, the invention encompasses the prodrug esters of the modified fatty acids herein. Examples of this type of drug in the market, nutrition, and clinical trial literature included Glaxo's Lovaza (esters blends of omega-3 fatty acids, EPA, dHa, and alpha-linolenic acid), Abbott's Omacor (esters of omega-3 fatty acid) and most fish oil supplements (DHA and EPA esters). In some aspects, the incorporation of the ester prodrugs into tissues or cells refers to the incorporation of the original modified PUFA, as it would be used as a body constituent.

In some embodiments, the stabilized compositions mimic naturally occurring fatty acids without changing their elemental composition. For example, the substituent may retain the chemical valence shell. Some embodiments include naturally occurring fatty acid mimetics and their ester prodrugs, which are chemically modified to be effective in preventing specific disease mechanisms, but are modified in a manner (such as isotopic substitution) that does not change the elemental composition of the material. For example, deuterium is a form of the same element hydrogen. In some aspects, these compounds maintain elemental composition and are stabilized against oxidation. Some compounds that are stabilized against oxidation are stabilized at sites sensitive to oxidation. Some compounds are stabilized against oxidation by heavy isotope substitution, then bis-allylic carbon hydrogen bonding, etc.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs can be protected against hydrogen extraction by further separating double bonds that activate the bis-allylic hydrogen, thus eliminating bis-allylic positions while retaining some fluidity of the bis-allylic sites. PUFAs as shown below. These PUFA mimetics do not have bis-allylic positions.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs can be protected against hydrogen extraction using valence II heteroatoms, thereby removing bis-allylic hydrogens as shown below. These PUFA mimetics also do not have bis-allylic hydrogens.

X = S: 10-hept-1 -enylsulfanyl acid X = S 10-(2-but-1 -enylsulfanyl acid

dec-9-enoic vimlsulfaml)-dec-9-enoic

X = O: 10-hept-1-emloxy-dec-9 acid X = O: 10-(2-but-1 -enryloxy

vimloxy)-dec-9-enoic

In a further embodiment, PUFA mimetics, ie compounds that are structurally similar to natural PUFAs but cannot be oxidized due to structural differences, can be used for the purposes mentioned above. Oxidation-prone bis-allylic sites of PUFAs can be protected against hydrogen extraction by di-methylation or halogenation, as shown below. The hydrogen atoms of the methyl groups may optionally be halogens, such as fluorine, or deuterium. These PUFA mimetics are dimethylated at bis-allylic sites.

In a further embodiment, the PUFA mimetics are dimethylated at bis-allylic sites, represented by the following compounds:

11,11-dimethyl-octadeca-9,12-dienoic acid 11,11,14,14-tetramethyl-octadeca-9,12,15-trienoic acid

R = H, C3H7- R1 = H ;alkyl; n = 1-5; m = 1- 12: X = F

R=H, C3H7; R=H;alkyl;n=1-5: m=1-12

In a further embodiment, bis-allylic sites of PUFAs prone to oxidation can be protected against hydrogen extraction by alkylation as shown below:

10-(1-hept-1-enyl-cyclopropyl) acid 10-{1-[2-(1-but-1-emt-cyclopropyl) acid

dec-9-enoic vinyl -cyclopropyl}-dec-9-erioic

In a further embodiment, cyclopropyl groups can be used in place of double bonds, thus giving the acids some fluidity while removing bis-allylic sites as shown below. These PUFA mimetics have cyclopropyl groups instead of double bonds.

8-[2-(2-pentyl- 8-{2- 2-{2-ethyl-cyclopropylmethyl) acid

cyclopropylmethyl-cyclopropyl cyclopropylmethyl[]-cyclopropyl}-octanoic acid

octanoic

R = H, C3H7; R ¹ _ H; I rent

In a further embodiment, 1,2-substituted cyclobutyl groups in the appropriate conformation may be used in place of double bonds, thus giving the acids some fluidity while removing bis-allylic sites as shown below. These PUFA mimetics have 1,2-cyclobutyl groups instead of double bonds.

8-[2-(2-pentyl-cyclobutylmethyl)- 8-{2-[2-(2-ethyl-cyclobutylmethyl)-cyclobutyl]-octanoic acid cyclobutylmethyl]-cyclobutyl}-octanoic acid

In a modification of the above embodiment of mimetics with 1,2-cyclobutyl groups in place of double bonds, 1,3-substituted cyclobutyl groups in the appropriate conformation may be used in place of double bonds, thus giving the acids some fluidity while bis-allylic sites are removed. The following PUFA mimetics have 1,3-cyclobutyl groups instead of double bonds.

8-[3-(3-pentyl-cyclobutylmethyl)-8-{3-3-(3-ethyl-cyclobutylmethyl)-cyclobutyl]-octanoic acid cyclobutylmethyl-cyclobutylj-octanoic acid

It is a well known principle in medicinal chemistry that certain functional groups are isosteric and/or bioisosteric with certain other functional groups. Bioisosteres are substituents or groups with similar physical or chemical properties that produce biological properties very similar to those of a chemical compound. For example, isosteres and/or bioisosteres for hydrogen include halogens such as fluorine; the isosteres and/or bioisosteres of alkenes include alkynes, phenyl rings, cyclopropyl rings, cyclobutyl rings, cyclopentyl rings, cyclohexyl rings, thioethers, and the like; isosteres and/or bioisosteres of carbonyls include sulfoxides, sulfones, thiocarbonyls, and the like; ester isosteres and/or bioisosteres include amides, sulfonic acid esters, sulfonamides, sulfinyl acid esters, sulfinylamines, and the like. Accordingly, PUFA mimetics also include compounds having isosteric and/or bioisosteric functional groups.

It is contemplated that it may be useful to formulate PUFA and/or PUFA mimetics as a prodrug for use in the invention. A prodrug is a drug substance that itself may have biological activity, but after administration, the prodrug is metabolized into a form that also exerts biological activity. Many different types of prodrugs are known and can be classified into two main types based on their cellular sites of metabolism. Type I prodrugs are those that are metabolized intracellularly, while type II prodrugs are those that are metabolized extracellularly. It is well known that carboxylic acids can be converted to esters and various other functional groups to improve pharmacokinetics, such as absorption, distribution, metabolism, and excretion. Esters are a well-known prodrug form of carboxylic acids formed by the condensation of an alcohol (or its chemical equivalent) with a carboxylic acid (or its chemical equivalent). In some embodiments, alcohols (or their chemical equivalents) for incorporation into PUFA prodrugs include alcohols or pharmaceutically acceptable chemicals that upon metabolism provide pharmaceutically acceptable alcohols. Such alcohols include, but are not limited to, propylene glycol, ethanol, isopropanol, 2-(2-ethoxyethoxy)ethanol (Transcutol®, Gattefosse, Westwood, NJ 07675), benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, or polyethylene glycol 400. ; polyoxyethylene castor oil derivatives (e.g., polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil (Cremophor®EL, BASF Corp.), polyoxyethyleneglycerol oxystearate (Cremophor®RH 40 (polyethylene glycol 40 hydrogenated castor oil) or Cremophor®RH 60 (polyoxyethylene glycol 60 hydrogenated castor oil), BASF Corp.)); saturated polyglycolyzed glycerides (eg, Gelucire® 35/10, Gelucire® 44/14, Gelucire® 46/07, Gelucire® 50/13, or Gelucire® 53/10, available from Gattefosse, Westwood, N.J. 07675); polyoxyethylene alkyl ethers (eg cetomacrogol 1000); polyoxyethylene stearates (eg, PEG-6 stearate, PEG-8 stearate, polyoxyl 40 NF stearate, polyoxyethyl 50 NF stearate, PEG-12 stearate, PEG-20 stearate, PEG-100 stearate, distearate of PEG-12, PEG-32 distearate, or PEG-150 distearate); ethyl oleate, isopropyl palmitate, isopropyl myristate; dimethyl isosorbide; N-methylpyrrolidinone; paraffin; cholesterol; lecithin; suppository bases; pharmaceutically acceptable waxes (eg, carnauba wax, yellow wax, white wax, microcrystalline wax, or emulsifying wax); pharmaceutically acceptable silicon fluids; sorbitan fatty acid esters (including sorbitan laurate, sorbitan oleate, sorbitan palmitate, or sorbitan stearate); pharmaceutically acceptable saturated fats or pharmaceutically acceptable saturated oils (for example, hydrogenated castor oil (glyceryl-tris-12-hydroxystearate), cetyl esters wax (a mixture of mainly C14-C18 saturated esters of C14-C18 saturated fatty acids having a melting range of about 43°-47°C), or glyceryl monostearate).

In some embodiments, the fatty acid prodrug is represented by the P-B ester, where the P moiety is a PUFA and the B moiety is a biologically acceptable molecule. Thus, cleavage of the P-B ester provides a PUFA and a biologically acceptable molecule. Said cleavage can be induced by acids, bases, oxidizing agents and/or reducing agents. Examples of biologically acceptable molecules include, but are not limited to, nutritional materials, peptides, amino acids, proteins, carbohydrates (including monosaccharides, disaccharides, polysaccharides, glycosaminoglycans, and oligosaccharides), nucleotides, nucleosides, lipids (including mono-, di-, and glycerols). tri-substituted, glycerophospholipids, sphingolipids and steroids).

In some embodiments, alcohols (or their chemical equivalent) for incorporation into PUFA prodrugs include alcohols with 1 to 50 carbon atoms ("C1-50 alcohols"), C1-45 alcohols, C1-40 alcohols, C1-50 alcohols, 35, C1-30 alcohols, C1-25 alcohols, C1-20 alcohols, C1-15 alcohols, C1-10 alcohols, C1-6 alcohols (wherever it appears herein, a numerical range such as "1-50" refers to refers to each integer in the given range, eg, "1 to 50 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 50 carbon atoms inclusive, although the present definition also covers the occurrence of the term "alkyl" where a numerical range is not designated). Said alcohols may be branched, unbranched, saturated, unsaturated, polyunsaturated and/or include one or more heteroatoms such as nitrogen, oxygen, sulfur, phosphorous, boron, silicone, fluorine, chlorine, bromine or iodine. Examples of alcohols include methyl, ethyl, propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, perfluoromethyl, perchloromethyl, perfluorotert-butyl, perchloro-tert-butyl and benzyl alcohols as well. of ether alcohols such as polyethylene glycols. In some embodiments, the alcohol contains a charged species. Said species can be anionic or cationic. In some embodiments, the species is a positively charged phosphorus atom. In other embodiments, the positively charged phosphorous atom is a phosphonium cation. In other embodiments, the charged species is a primary, secondary, tertiary, or quaternary ammonium cation.

In some embodiments, alcohols (or their chemical equivalents) for incorporation into PUFA prodrugs include polyols such as diols, triols, tetraols, pentaols, etc. Examples of polyols include ethylene glycol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, methylpropanediol, ethoxydiglycol, hexylene glycol, dipropylene glycol, glycerol, and carbohydrates. Esters formed from polyols and PUFAs can be monoesters, diesters, triesters, etc. In some embodiments, the multiple esterified polyols are esterified with the same PUFAs. In other embodiments, the multiple esterified polyols are esterified with different PUFAs. In some embodiments, the different PUFAs are stabilized in the same way. In other embodiments, the different PUFAs are stabilized different ways (such as deuterium substitution in one PUFA and 13C substitution in another PUFA). In some embodiments, one or more PUFAs are an omega-3 fatty acid and one or more PUFAs are an omega-6 fatty acid.

It is also contemplated that it may be useful to formulate PUFAs and/or PUFA mimetics and/or PUFA prodrugs as salts for use in the invention. For example, the use of salt formation as a means of tailoring the properties of pharmaceutical compounds is well known. See Stahl et al., Handbook of pharmaceutical salts: Properties, selection and use (2002) Weinheim/Zurich: Wiley-VCH/VHCA; Gould, Salt selection for basic drugs, Int. J. Pharm. (1986), 33:201-217. Salt formation can be used to increase or decrease solubility, improve stability or toxicity, and reduce hygroscopicity of a drug.

The formulation of PUFAs and/or PUFA mimetics and/or PUFA prodrugs as salts includes, but is not limited to, the use of basic inorganic salt-forming agents, basic organic salt-forming agents, and salt-forming agents containing both acidic and basic functional groups. Various inorganic bases useful for forming salts include, but are not limited to, alkali metal salts such as lithium, sodium, potassium, rubidium, cesium, and francium salts, and alkaline earth metal salts such as beryllium, magnesium, calcium, strontium, barium and radium, and metals such as aluminum. These inorganic bases may further include counterions such as carbonates, hydrogen carbonates, sulfates, hydrogen sulfates, sulfites, hydrogen sulfites, phosphates, hydrogen phosphates, dihydrogen phosphates, phosphites, hydrogen phosphites, hydroxides, oxides, sulfides, alkoxides such as methoxy, ethoxy, and t-butoxide and the like. Various organic bases useful for forming salts include, but are not limited to, amino acids, basic amino acids such as arginine, lysine, ornithine and the like, ammonia, alkylamines such as methylamine, ethylamine, dimethylamine, diethylamine, trimethylamine, triethylamine and the like, heterocyclic amines such as pyridine, picoline and the like, alkanolamines such as ethanolamine, diethanolamine, triethanolamine and the like, diethylaminoethanol, dimethylaminoethanol, N-methylglucamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, ethylenediamine, piperazine, choline, trolamine, imidazole, diolamine, betaine, tromethamine, meglumine, chloroprocaine, procaine and the like.

PUFA salt and/or PUFA mimetic and/or PUFA prodrug formulations include, but are not limited to, pharmaceutically acceptable basic inorganic salts, basic organic salts, and/or organic compounds having both acidic and basic functional groups. Pharmaceutically acceptable salts are well known in the art and include many of the organic and inorganic bases mentioned above. Pharmaceutically acceptable salts further include salts and salt-forming agents found in drugs approved by the Food and Drug Administration and foreign regulatory agencies. Pharmaceutically acceptable organic cations for incorporation include, but are not limited to, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, benethamine, clemizole, diethylamine, piperazine, and tromethamine. Pharmaceutically acceptable metal cations for incorporation include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, barium, and bismuth. Additional salt-forming agents include, but are not limited to, arginine, betaine, carnitine, diethylamine, L-glutamine, 2-(4-imidazolyl)ethylamine, isobutanolamine, lysine, N-methylpiperazine, morpholine, and theobromine.

In addition, there are several lists of pharmaceutically approved counterions. See Bighley et al., Salt forms of drugs and absorption. 1996 In: Swarbrick J. et al. eds. Encyclopaedia of pharmaceutical technology, vol. 13 New York: Marcel Dekker, Inc. pp. 453-499; Gould, P.L., Int. J. Pharm. 1986, 33, 201-217; Berge, J. Pharm. Sci. 1977, 66, 119; Heinrich Stahl P., Wermuch C.G. (eds), Handbook of Pharmaceutical Salts, IUPAC, 2002; Stahl et al., Handbook of pharmaceutical salts: Properties, selection and use (2002) Weinheim/Zurich: Wiley-VCH/VHCA, all of which are incorporated herein by reference.

Various inorganic bases useful for forming salts include, but are not limited to, alkali metal salts such as lithium, sodium, potassium, rubidium, cesium, and francium salts, and alkaline earth metal salts such as beryllium, magnesium, calcium, strontium, barium and radium, and metals such as aluminum. These inorganic bases may further include counterions such as carbonates, hydrogen carbonates, sulfates, hydrogen sulfates, sulfites, hydrogen sulfites, phosphates, hydrogen phosphates, dihydrogen phosphates, phosphites, hydrogen phosphites, hydroxides, oxides, sulfides, alkoxides such as methoxy, ethoxy, and t-butoxide and the like.

Various organic bases useful for forming salts include, but are not limited to, amino acids; basic amino acids such as arginine, lysine, ornithine and the like; ammonia; ammonium hydroxide; alkylamines such as methylamine, ethylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, and the like; heterocyclic amines such as pyridine, picoline, and the like; alkanolamines such as ethanolamine, diethanolamine, triethanolamine and the like, diethylaminoethanol, dimethylaminoethanol; N-methylglucamine; dicyclohexylamine; N,N'-dibenzylethylenediamine; ethylenediamine; piperazine; hill; trolamine; imidazole; diolamine; betaine; tromethamine; meglumine; Chloroprocaine; procaine; and the like.

In some embodiments, it is not necessary to replace all non-isotopically modified PUFAs, such as non-deuterated PUFAs, with isotopically modified PUFAs, such as deuterated PUFAs. In some embodiments, it is preferable to have enough isotopically modified PUFAs, such as D-PUFAs, in the membrane to prevent unmodified PUFAs, such as H-PUFAs, from sustaining an autoxidation chain reaction. During autoxidation, when a PUFA is oxidized and there is an unoxidized PUFA nearby, the unoxidized PUFA can be oxidized by the oxidized PUFA. This may also be referred to as autoxidation. In some cases, if there is a low concentration, for example, H-PUFA "diluted" in the membrane with D-PUFA, this oxidation cycle can break down due to the distance separating the H-PUFAs. In some embodiments, the concentration of isotopically modified PUFAs is present in an amount sufficient to support the autoxidation chain reaction. To break the autoxidation chain reaction, for example, 1-60%, 5-50% or 15-35% of the total molecules of the same type are in the membrane. This can be measured by IRMS (isotope ratio mass spectrometry).

Another aspect of the invention provides a dietary, supplemental or pharmaceutical composition of the active compounds. In some embodiments, the dietary, supplemental, or pharmaceutical composition may comprise a salt of the active compound.

Co-administration

In some embodiments, one or more stabilized substances, such as PUFAs, are administered before, at the same time as, or after the administration of an additional therapeutic agent useful for treating or inhibiting certain diseases, or reducing side effects associated with certain diseases. In some embodiments, the PUFAs and the additional therapeutic agent are useful for treating the same disease.

In some embodiments, the compounds described herein are administered in combination. For example, in some embodiments, two, three, four, and/or five or more stabilized compounds are administered together. In some embodiments, the stabilized compounds are administered in approximately similar amounts. In other embodiments, the stabilized compounds are administered in different amounts. For example, any two or more compounds in a mixture may represent about 1% to about 99% of a mixture, about 5% to about 95% of a mixture, about 10% to about 90% of a mixture, about 15% to about 85% of a mixture, about 20% to about 80% of a mixture, about 25% to about 75% of a mixture, about 30% to about 70% of a mixture, about 35% to about 65% of a mixture mixture, about 40% to about 60% of a mixture, about 40% to about 60% of a mixture, about 45% to about 55% of a mixture, and/or about 50% of a mixture . In other embodiments, any two or more compounds in a mixture can represent approximately: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a mix, or a range limited by any two of the numbers listed above.

Although antioxidants cannot cancel the negative effects of PUFA peroxidation due to the stochastic nature of the process and the stability of PUFA peroxidation products (reactive carbonyls) to antioxidant treatment, co-administration of antioxidants with oxidation-resistant compositions, such as those described herein, may be beneficial for the treatment of disorders related to oxidative stress.

Certain antioxidants considered useful for coadministration include the following: vitamins, such as vitamin C and vitamin E; glutathione, lipoic acid, uric acid, sulforaphane carotenes, lycopene, lutein, anthocyanins, oxalic acid, phytic acid, tannins, coenzyme Q, melatonin, tocopherols, tocotrienols, polyphenols including resveratrol, flavonoids, selenium, eugenol, idebenone, mitoquinone, mitoquinol , ubiquinone, Szeto-Schiller peptides and antioxidants targeting the mitochondria. When not explicitly mentioned, quinone derivatives of the aforementioned antioxidants are also contemplated as being useful for co-administration.

In some embodiments, the stabilized compounds are administered with compounds that up-regulate antioxidant genes. In other embodiments, the stabilized compounds are administered with compounds that affect signaling pathways, such as the Keap1/Nrf2/ARE signaling pathway, resulting in the production of anti-inflammatory and/or antioxidant proteins, such as heme oxygenase-1 (HO -one). In some embodiments, the stabilized compounds are administered with antioxidant inflammation modulators. Antioxidant modulators of inflammation suppress prooxidant and/or proinflammatory transcription factors. In some embodiments, the antioxidant inflammation modulators are activators of the transcription factor Nrf2. Activation of Nrf2 promotes the up-regulation of antioxidant, detoxifying and anti-inflammatory genes. In other embodiments, the antioxidant inflammation modulators suppress NF- ^kB . In some embodiments, the antioxidant inflammation modulators suppress STAT3. In other embodiments, the stabilized compounds are administered with compounds that affect histone deacetylase activity. In some embodiments, the stabilized compounds are administered with compounds that bind antioxidant response elements (AREs). In other embodiments, the stabilized compounds are administered with methyl-bardoxolone (2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid methyl ester) as the antioxidant modulator of inflammation. In some embodiments, the antioxidant inflammation modulator is 2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid, or a pharmaceutically acceptable ester thereof. In other embodiments, the antioxidant inflammation modulator is a 2-cyano-3,12-dioxooleano-1,9(11)-dien-28-oic acid amide. In some embodiments, the antioxidant inflammation modulator is a triterpenoid. In other embodiments, the antioxidant inflammation modulator is selected from the following compounds:

Additional antioxidants believed to be useful in coadministration therapies include the compounds disclosed in US Patent Nos. 6,331,532; 7,179,928; 7,232,809; 7,888,334; 7,888,335; 7,432,305; 7,470,798; and 7,514,461; and US Patent Application Nos. 20020052342; 20030069208; 20040106579; 20050043553; 20050245487; 20060229278; 20070238709; 20070270381; 20080161267; 20080275005; 20090258841; 20100029706; and 20110046219; wherein the compounds described herein are incorporated by reference.

In addition, it is contemplated that co-administration of antioxidants could take the form of consuming foods known to have higher levels of beneficial antioxidants. Such foods include both regular foods and "superfoods" that contain antioxidants. These foods include fruits, vegetables, and other foods such as strawberries, blackcurrants, blackberries, oranges, blueberries, pomegranates, tea, coffee, olive oil, chocolate, cinnamon, herbs, red wine, cereal grains, eggs, meat, legumes, nuts, spinach, turnip, rhubarb, cocoa beans, corn, beans, cabbage and the like.

In some embodiments, one or more stabilized substances, such as one or more PUFAs, are administered before, at the same time as, or after the administration of an additional therapeutic agent useful for treating an ophthalmic disorder, such as optic neuropathy. In some embodiments, the optic neuropathy is glaucoma. Compounds useful for treating or inhibiting glaucoma, or reducing the side effects of glaucoma, are known to those of skill in the art. Such compounds include cholinergic agonists; anticholinesterase agents; muscarinic antagonists; sympathomimetic agents; α- and p-andrenergic antagonists; carbonic anhydrase inhibitors; prostaglandin analogs; and marijuana.

In some embodiments, one or more stabilized substances, such as PUFAs, are administered before, at the same time as, or after surgery to treat or inhibit an ophthalmic disorder, such as optic neuropathy. In some embodiments, the optic neuropathy is glaucoma. In some embodiments, the surgical intervention is laser trabeculoplasty; argon laser trabeculoplasty; selective laser trabeculoplasty; iridotomy; peripheral laser iridotomy; iridectomy; sclerotomy; anterior sclerotomy; trabeculectomy; viscocanalostomy; goniotomy; tube bypass surgery; goniocurretage; cyclodialysis; cyclogoniotomy; canaloplasty; cyclocryotherapy; cyclophotocoagulation; ciliary body ablation; cyclophotoablation; cyclophototherapy; cyclodiathermy; or cycloelectrolysis.

Administration and additional formulations:

It is well known that triglycerides are the main components of vegetable oils and animal fats. It is also known that a triglyceride is an ester compound derived from glycerol and three fatty acids. Triglycerides are metabolized by enzymes such as lipases that hydrolyze ester bonds to release fatty acids and glycerol. In fact, this metabolism releases fatty acids that can then be absorbed by cells through a fatty acid carrier protein. It is contemplated that PUFAs and PUFA mimetics that are useful in the treatment of various diseases can be incorporated into fats such as triglycerides, diglycerides, and/or monoglycerides for administration to a patient.

Administration of PUFAs, PUFA mimetics, PUFA prodrugs, and PUFA-containing triglycerides and/or PUFA mimetics could be through a modified diet. Alternatively, PUFAs, PUFA mimetics, PUFA prodrugs, and PUFA-containing triglycerides and/or PUFA mimetics may be administered as foods or food supplements, alone or as complexes with 'carriers', including, but not limited to, complexes with albumin.

Other methods of administering the enhanced PUFAs or their precursors may also be employed, such as the methods typically used for drug delivery and drug delivery. These methods include, but are not limited to, peroral administration, topical administration, transmucosal administration such such as nasal administration, nasal administration through a cribriform plate, intravenous administration, subcutaneous administration, inhalation, or via eye drops.

Targeted delivery methods and sustained release methods may also be employed, including, but not limited to, the liposome delivery method.

It is contemplated that the isotopically modified compounds described herein may be administered over a period of time, in which the cells and tissues of the subject will contain increasing levels of isotopically modified compounds over the course of time that the compounds are administered. For example, the compounds can be administered for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days. , 1 month, 2 months, 3 months, 4 months, five months, 6 months, 9 months, 1 year, 2 years, 5 years or 10 years, or a range limited by any two of the above numbers. In some embodiments, the compounds are administered for about 2 months.

Compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, dragees, lozenges, aqueous or oily suspensions, oil-in-water emulsions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Such compositions may contain excipients such as bulking agents, solubilizing agents, flavor-masking agents, stabilizers, coloring agents, preservatives, and other agents known to those of ordinary skill in the art of pharmaceutical formulation. Additionally, oral forms can include foods or food supplements containing the compounds described herein. In some embodiments, supplements can be tailored so that one type of PUFA, such as omega-3 or omega-6 fatty acids, can be added to foods or used as a supplement depending on the dominant fat that the food contains. or the subject's diet. Furthermore, the compositions can be tailored depending on the disease to be treated. For example, an LDL-related condition may require more D-linoleic acid because cardiolipin, which is made from linoleic acid, is oxidized. In other embodiments, such as retinal disease and neurological/CNS conditions, more omega-3 fatty acids such as D-linolenic acid may be required, because D-omega-3 fatty acids are more relevant to treating these diseases. In some respects, when the disease is associated with HNE, then D-omega-6 fatty acids should be prescribed, whereas for HHE, D-omega-3 fatty acids should be prescribed.

The compositions may also be suitable for administration by topical application, as a spray, cream, ointment, lotion, or as a component or additive to a patch, bandage, or wound dressing. In addition, the compound can be delivered to the disease site by mechanical means or targeted to the disease site through the use of systemically targeted technologies such as liposomes (with or without chemical modification that provides them with affinity for diseased tissue), antibodies, aptamers, lectins, or chemical ligands such as albumin, with an affinity for aspects of diseased tissue that are less abundant or not present in normal tissue. In some aspects, the topical application of cosmetics may include the use of a carrier that is an isotopically modified compound or mimetic described herein for administration through the skin, such as by means of a patch. Eye disorders can be treated with eye drops.

A pharmaceutical composition may also be in a form suitable for administration by injection. Said compositions may be in the form of a solution, suspension or emulsion. Such compositions may include stabilizing agents, antimicrobial agents, or other materials to enhance the function of the drug. Some aspects of the invention also encompass dry or desiccated forms of the compounds that can be readily formed or reconstituted into a solution, suspension, or emulsion suitable for administration by injection, or for oral or topical use. Administration by injection may be suitable for systemic administration and also local administration, such as injection into the eye, to treat disorders related to the eye.

Dose

In some embodiments, the compounds are dosed at about 0.01 mg/kg to about 1000 mg/kg, about 0.1 mg/kg to about 100 mg/kg, and/or about 1 mg/kg to about 10 mg/kg. . In other embodiments, the compounds are dosed at approximately: 0.01, 0.1, 1.0, 5.0, 10, 25, 50, 75, 100, 150, 200, 300, 400, 500 and/or 1000 mg/kg. In some embodiments, the compounds are dosed orally with a morning, afternoon, and/or evening meal. In some embodiments, 0.25 g, 0.5 g, 0.75 g, 1 g, 1.25 g, 1.5 g, 1.75 g, 2 g, 2.5 g, 3 g are administered daily. , 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 9g, 10 or 15g, or a range limited by any two of the numbers listed above. In other embodiments, the amounts mentioned above are dosed in a single meal. In some embodiments, the desired daily dose is administered over two or more meals. In some embodiments, 1 g, 2 g, 4 g, or 8 g per day is administered.

examples

Certain compounds useful for the methods described herein can be prepared according to known methodologies or ready modifications of known methodologies. For example, deuterium-substituted cis-9-octadecenoate-14,14,15,15,17,18-d6 and methyl cis-9,cis-12-octadecadienoate-12,13-d2 and triglycerides of these can be prepared. variants. as previously described in Adlof et al., J. Labeled Compd. Radiopharm. (1978), vol. fifteen, pp.97-104; Adlof et al., J. Am. Oil. Chem. Soc. (1993), Vol. 70, pp.817-819; and Emken et al., Lipids (1999), vol. 34, pp.785-791.

Additional compounds useful for the methods described herein can be prepared as described in WO 2012/148926; WO 2012/148930; WO 2012/148927; and WO 2012/148929.

One skilled in the art will recognize that carboxylic acids can be readily converted to further derivatives, such as esters, by treatment with known reagents and the desired alcohol as described in Scheme 1.

Scheme 1. General preparation of esters A-D

P U F A -------- Alcohol/Polyalcohol RUFA -------- Polyalcohol -------- RUFA

RUFA

PUFA-------- Polyol -------- RUFA

PU FA-------- Polyol -------- PUFA

PUFA

cPUFA

D

General procedure for compound A. Thionyl chloride (2 equivalents) is added slowly to a solution of polyunsaturated fatty acid (1 equivalent) in CHCh. The reaction mixture is heated under reflux for 1 hour, then allowed to cool to room temperature and the solvent evaporated under reduced pressure to give the polyunsaturated fatty acid carboxylic acid chloride derivative. The carboxylic acid chloride derivative is then dissolved in anhydrous pyridine and the alcohol (1 equivalent) dissolved in pyridine is slowly added (note that the order of addition is reversed when the alcohol is a polyol). After addition is complete, the reaction mixture is allowed to stir at room temperature for 24 h. The solvent is then removed under reduced pressure and the crude product is purified by column chromatography to give Compound A.

General procedure for compound B. Thionyl chloride (2 equivalents) is added slowly to a solution of polyunsaturated fatty acid (1 equivalent) in CHCh. The reaction mixture is heated under reflux for 1 hour, then allowed to cool to room temperature and the solvent evaporated under reduced pressure to give the polyunsaturated fatty acid carboxylic acid chloride derivative. Next, the carboxylic acid chloride derivative is dissolved in anhydrous pyridine, and the alcohol (Compound A, 1 equivalent) dissolved in pyridine is added slowly. After addition is complete, the reaction mixture is allowed to stir at room temperature for 24 h. The solvent is then removed under reduced pressure and the crude product is purified by column chromatography to provide Compound B.

General procedure for compound C. Thionyl chloride (2 equivalents) is added slowly to a solution of polyunsaturated fatty acid (1 equivalent) in CHCl3. The reaction mixture is heated under reflux for 1 hour, then allowed to cool to room temperature and the solvent evaporated under reduced pressure to give the polyunsaturated fatty acid carboxylic acid chloride derivative. Next, the carboxylic acid chloride derivative is dissolved in anhydrous pyridine, and the alcohol (Compound B, 1 equivalent) dissolved in pyridine is added slowly. After addition is complete, the reaction mixture is allowed to stir at room temperature for 24 h. The solvent is then removed under reduced pressure and the crude product is purified by column chromatography to provide Compound C.

General procedure for compound D. Thionyl chloride (2 equivalents) is added slowly to a solution of polyunsaturated fatty acid (1 equivalent) in CHCl3. The reaction mixture is heated under reflux for 1 hour, then allowed to cool to room temperature and the solvent evaporated under reduced pressure to give the polyunsaturated fatty acid carboxylic acid chloride derivative. Next, the carboxylic acid chloride derivative (4 equivalents) is dissolved in anhydrous pyridine, and the alcohol (1 equivalent) dissolved in pyridine is added slowly. After addition is complete, the reaction mixture is allowed to stir at room temperature for 24 h. The solvent is then removed under reduced pressure and the crude product is purified by column chromatography to provide Compound D.

Example 1. Effects on yeast without Q

Yeast without Q (coq mutants) provide an ideal system to assess in vivo autoxidation of fatty acids.

Coenzyme Q (ubiquinone or Q) acts as a small lipophilic antioxidant and as an electron shuttle in the respiratory chain of the inner mitochondrial membrane. Ten S. cerevisiae genes (COQ1-COQ10) are required for coenzyme Q biosynthesis and function, and clearance of any results in respiratory failure (T ran UC, Clarke CF. Mitochondrion 2007; 7S,S62). Yeast coq mutants were shown to be exquisitely sensitive to PUFA autoxidation products (Do t Q et al, PNAS USA 1996; 93:7534-7539; Poon WW, Do TQ, Marbois b N, Clarke CF. Mol Aspects Med. 1997;18, s121). Although S. cerevisiae do not produce PUFAs (Paltauf F, Daum G. Meth. Enzymol. 1992; 209: 514-522), they can use PUFAs when provided exogenously, allowing their content to be manipulated (Paltauf F, Daum G. Meth Enzymol 1992;209:514-522). Less than 1% of the non-Q yeast mutants (coq2, coq3, and coq5) are viable after four hours treatment with linolenic acid (Do TQ et al, PNAS USA 1996; 93:7534-7539; Poon Ww, Do TQ, Marbois BN, Clarke CF. Mol. Aspects Med. 1997;18, s121). In contrast, 70% of wild-type cells (parental genetic background is strain W303-1B) subjected to this treatment remain viable. Yeasts without Q are also hypersensitive to other easily autooxidized PUFAs (such as arachidonic acid), but behave the same as the wild-type parent strain when treated with monounsaturated oleic acid (Do TQ et al, PNAS USA 1996; 93 :7534-7539). The hypersensitivity of Q-less yeast mutants is not a secondary effect of inability to breathe, because cor1 or atp2 mutant yeast (lacking the bcl1o complex of ATP synthase, respectively) show wild-type resistance to PUFA treatment. (Dot Q et al, PNAS USA 1996; 93:7534-7539; Poon WW, Do TQ, Marbois BN, Clarke CF. Mol. Aspects Med. 1997; 18, s121).

A plate dilution assay can be used to assess sensitivity to PUFAs. This assay is performed by plating serial five-fold dilutions of aliquots onto YPD plating medium. The sensitivity of the different strains is observed by visual inspection of the cell density at each point.

Cell types representative of oxidation-related disorders can also be evaluated. For example, said cells are maintained in medium containing hydrogenated compounds (control) or isotopically modified/stabilized compounds (20 pM) for 72 hours. PUFA incorporation into cells is monitored by GC. Cells are then treated with paraquat (PQ; 500 pM), a common compound that generates oxidative stress. For measurement of survival, cells are counted using a hemocytometer and trypan blue exclusion method.

Alternatively, experiments can be performed in which cells are treated with various concentrations of steroids (glucocorticoids, corticosteroids, etc.) with PUFA pre- and post-treatment in the presence of an oxidative stress-generating compound, such as paraquat. For measurement of survival, cells are counted using a hemocytometer and trypan blue exclusion method.

Example 2. Evaluation of Neuroblastoma Cells

Dhcr7-deficient Neuro2a neuroblastoma cells were treated with D2-linolenic acid, and non-deuterated linoleic acid ("Lin") was used in a control experiment. The level of Dhcr7 gene expression was decreased by RNA interference, leading to accumulation of 7-DHC in cells. 7-DHC is 1-2 orders of magnitude more oxidizable than linoleic acid, so it can easily initiate lipid peroxidation (See Yin et al., "Free Radical Lipid Peroxidation: Mechanisms and Analysis", Chem Rev. (2011 ), 111(10), 5944-5972). Non-deuterated Lin (at a level of 20 micromolar) was found to substantially increase the levels of oxysterols associated with free radical-induced oxidation, whereas D2-Lin at the same concentration did not induce oxidative stress, judging by the low corresponding oxysterol levels.

Example 3. Histopathological studies

Histopathological studies can also be performed for the compounds described herein. Microscopic changes are coded by the most specific topographical and morphological diagnosis, and the terminology manuals of the Systematized Nomenclature of Medicine (SNOMED) and the Toxicology Data Management System (TDMS) of the National Toxicology Program are used as guidelines. Data is recorded in Labcat® Histopathology module 4.30. A four-step rating system (minimal, minor, moderate, and strong) is used to define the changes that can be rated.

Male C57BL6 mice are dosed orally on the PUFA diet on study days 1 to 14, and are necropsied on study day 15. Group 1 may consist of 4 mice and receive isotopically unmodified polyunsaturated substances. . Group 2 may consist of 5 mice and receive isotopically modified polyunsaturated substances. On day 8 of the study, all mice received intraperitoneal (IP) saline. Complete sets of protocol-specified tissues [liver (3 to 7 sections), lungs with bronchi (2 to 5 lobes), spleen, heart, and kidneys] from all delivered mice are examined histopathologically for differences.

Example 4. Evaluation of tissue-specific isotope incorporation

Incorporation of tissue-specific isotopes can also be determined. WT mice are housed at 12 animals (males separated from females) per cage and fed for 90 days ad libitum (typically 5-6 g/day) with the AIN 93 diet, as pellets, with 6% of total fat. About 10% of that total fat is composed of isotopically modified compounds or unmodified compounds (control group). Animals are sacrificed, various organs (such as eyes) harvested and stored at low temperature prior to analysis without the use of preservatives. Lipid fractions are separated, pretreated and analyzed by LC-MS according to standard protocols. See Shchepinov et al. Toxicology Letters 207 (2011) 97-103. Furthermore, experiments can use transgenic mice whose genome comprises a homozygous alteration in a Vav2 and/or Vav3 gene but not an alteration in a Vav1 gene as a model for evaluating an optic neuropathy such as glaucoma.

Example 5: Model for testing incorporation in the eyes

Isotope ratio mass spectrometry can be used to confirm the incorporation of test compounds into the phospholipid membranes of various tissues, such as eyes, and specific components thereof. When test compounds are administered via dietary supplements, uptake into the eyes and specific components can be monitored using an isotope ratio mass spectrometry technique that will allow the overall increase in deuterium composition in lipid membranes to be measured. thus informing about the incorporation of test compounds, and any other PUFA derived from these compounds. Using this method, substantial absorption of test compounds in the eyes and specific components can be detected. For example, mice are supplemented with test compounds (0.01, 0.1, 1.0, 10.0, and 100 mg/kg of) or unmodified compounds (0.01, 0.1, 1.0 , 10.0, and 100 mg/kg) as the sole source of said compounds for 6 days, are acutely exposed to a known oxidant or saline vehicle, and continue on the same diet for an additional 6 days. The animals are sacrificed and the eyes are harvested, compartmentalized as desired and converted into homogenized samples from control mice and mice treated with test compound for isotope content analysis.

Example 6: efficacy against glaucoma

Glaucoma cells from human donor eyes are cultured according to known methods and exposed to D-PUFA (0.01, 0.1, 1.0, 10.0, 100 and 1000 pM D2-LA, D4- ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUfA (0.01, 0.1, 1.0, 10.0, 100 and 1000 pM La, ALA and 1: 1 from both LA and ALA). At various time points (24, 28 and 72 hours), digital images are taken and analyzed for lipofuscin and pigmentation according to known methods. The deuterated polyunsaturated fatty acid or ester thereof is expected to enhance the growth of retinal ganglion cells.

3-month-old female C57BL/6 mice (n=20) are used as a normal model. 8-month-old DBA/2J female mice (n=20) are used as a glaucoma mouse model. One eye of each animal is treated and the other eye serves as a control (for both normal and glaucoma models). DBA/2J mice spontaneously develop essential iris atrophy, pigment dispersion, and glaucomatous changes. Intraocular pressure (IOP) rises in most mice by the age of 9 months, which may be followed by ganglion cell loss, optic nerve atrophy, and optic nerve cupping. As glaucoma develops, ganglion cell loss and mild cupping of the optic nerve are present in some animals by 11 months and in most mice by age 22 months. T

Consequently, D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg of D2-LA, D4-ALA, and 1:1 combinations of both D2-LA and D4-ALA ) or H-PUfA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg LA, Al a and 1:1 combinations of both LA and ALA) are administered daily to mice having glaucoma . Over a twenty week period, the rats are examined for IOP, retinal ganglion cells and other indicators of the glaucoma condition. At the end of the trial, the rats are sacrificed, their eyes dissected and further examined for IOP, retinal ganglion cells and other indicators of the glaucoma condition. Deuterated polyunsaturated fatty acid or ester thereof lowers IOP, increases retinal ganglion cell growth and relieves glaucoma conditions.

Example 7: Efficacy against nonhereditary optic neuropathy

Mice having nonhereditary optic neuropathy are raised on a D-PUFA diet (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA, D4-ALA, and 1: 1 of both D2-LA and D4 -ALA) or H-PUfA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg of LA, ALA and 1:1 combinations of both LA and of ALA). After 1, 3, 5 and 7 weeks, the mice are sacrificed, their eyes are dissected and the retinas and oculars are combined and homogenized with ethanol. The homogenate is centrifuged, the supernatant is removed and the supernatant is analyzed for A2E-lipofuscin by HPLC. Deuterated polyunsaturated fatty acid or ester thereof is expected to reduce optic nerve swelling, reduce myelin loss.

In mice having nonhereditary optic neuropathy, deuterated polyunsaturated fatty acid or ester thereof, such as D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA, D4 -ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUfA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg LA, ALA and combinations 1 :1 of both LA and ALA) are administered to ocular or adnexal tissue. After 1, 3, 5, and 7 weeks, the mice are sacrificed, their eyes dissected, and the retinas and eyewashes are combined and homogenized with ethanol. The homogenate is centrifuged, the supernatant is removed, and the supernatant is analyzed for myelin cell count and other indicators of optic neuropathy. The deuterated polyunsaturated fatty acid or ester thereof reduces optic nerve swelling and reduces myelin loss.

Example 8: Efficacy against steroid-induced oxidative stress or steroid-induced oxidation-related disorder

Mice having steroid-induced oxidative stress or steroid-induced oxidation-related disorder are raised on a diet of deuterated polyunsaturated fatty acid or ester thereof as D-PUFA (0.01,0.1, 1.0, 10.0 and 100 mg/kg of D2-LA, D4-ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg of LA, ALA and 1:1 combinations of both LA and ALA). After 1, 3, 5, and 7 weeks, the mice are sacrificed, their eyes dissected, and the retinas and eyewashes are combined and homogenized with ethanol. The homogenate is centrifuged, the supernatant is removed and the supernatant is analyzed for A2E-lipofuscin by HPLC. Deuterated polyunsaturated fatty acid or ester thereof reduces myelin loss and reduces oxidative stress in ocular tissue.

In mice having steroid-induced oxidative stress or steroid-induced oxidation-related disorder, deuterated polyunsaturated fatty acid or ester thereof as D-PUFA (0.1, 0.1, 1.0, 10.0 and 100 mg/kg of D2-LA, D4-ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01,0.1, 1.0, 10.0 and 100 mg /kg of LA, ALA, and 1:1 combinations of both LA and ALA) are administered to the ocular or adnexal tissue of mice. After 1, 3, 5, and 7 weeks, the mice are sacrificed, their eyes dissected, and the retinas and eyewashes are combined and homogenized with ethanol. The homogenate is centrifuged, the supernatant is removed and the supernatant is analyzed for A2E-lipofuscin by HPLC. Deuterated polyunsaturated fatty acid or ester thereof reduces myelin loss and reduces oxidative stress in ocular tissue.

Example 9: Treatment of patients with glaucoma

Glaucoma patients are treated with deuterated polyunsaturated fatty acid or ester thereof, including D-PUFA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg of D2-LA, D4-ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01,0.1, 1.0, 10.0 and 100 mg/kg LA, ALA and 1:1 combinations of both of LA and ALA). After 1, 3, 5, and 7 weeks, patients are examined for iris atrophy, pigment dispersion, glaucomatous changes, intraocular pressure changes, and ganglion cell count. Deuterated polyunsaturated fatty acid or ester thereof is expected to reduce intraocular pressure change, increase ganglion cell count and improve other indicators of glaucoma.

Example 10: Treatment of patients with nonhereditary optic neuropathy

Patients with nonhereditary optic neuropathy are treated with deuterated polyunsaturated fatty acid or an ester thereof, including D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA , D4-ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUfA (0.01, 0.1, 1.0, 10.0 and 100 mg/kg LA, ALA and 1:1 combinations of both LA and ALA). After 1, 3, 5, and 7 weeks, patients are examined for iris atrophy, pigment dispersion, glaucomatous changes, intraocular pressure changes, myelin cell count, and other indicators of optic neuropathy. Deuterated polyunsaturated fatty acid or ester thereof is expected to reduce intraocular pressure change, increase myelin cell count and improve optic neuropathy conditions.

Example 11: Treatment of Patients Having Steroid-Induced Oxidative Stress or Steroid-Induced Oxidation-Related Disorder

Patients who have steroid-induced oxidative stress or a steroid-induced oxidation-related disorder are treated with deuterated polyunsaturated fatty acid or an ester thereof, including D-PUFA (0.01,0.1, 1.0, 10.0 and 100 mg/kg of D2-LA, D4-ALA and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01,0.1, 1.0, 10.0 and 100 mg /kg of LA, ALA and 1:1 combinations of both LA and ALA). After 1, 3, 5, and 7 weeks, patients are examined for iris atrophy, pigment dispersion, glaucomatous changes, changes in intraocular pressure, and other indicators of oxidative stress. The deuterated polyunsaturated fatty acid or ester thereof is expected to reduce optical oxidative stress.

Claims (14)

1. A polyunsaturated substance for use in a method of treating glaucoma, steroid-induced cataracts, steroid-induced myopathy, or Smith-Lemli-Opitz syndrome (SLOS), wherein the method comprises: identifying a patient as having glaucoma, steroid-induced cataract, steroid-induced myopathy, or Smith-Lemli-Opitz syndrome (SLOS) and requiring treatment; repeatedly administering a polyunsaturated substance to the patient or the patient's tissue, wherein the polyunsaturated substance is isotopically modified at one or more positions; wherein after said administration, the polyunsaturated substance is incorporated into the patient's body or the patient's tissue; wherein the polyunsaturated substance is a fatty acid, a fatty acid ester or a salt thereof. 2. The polyunsaturated substance for use according to claim 1, wherein the polyunsaturated substance is isotopically modified at one or more bis-allylic positions. 3. The polyunsaturated substance for use according to claim 1 or 2, wherein the polyunsaturated substance is chemically modified with at least one 13C atom or at least one deuterium atom in the one or more bis-allylic positions, and in wherein the at least one 13C atom or the at least one deuterium atom is present at a level significantly higher than the naturally occurring abundance level of said isotope. 4. The polyunsaturated substance for use according to any one of claims 1-3, wherein the polyunsaturated substance having one or more deuteriums has a structure of formula (6)

in which: R is H or C3H7; R1 is H, alkyl or cation; m is 1-10; n is 1-6; p is 1-10; Y1 through Yn is independently H or D; Y X1 through Xm is independently H or D. 5. The polyunsaturated substance for use according to any one of claims 1-3, wherein the polyunsaturated substance having one or more deuteriums has a structure of formula (2)

R is H or C3H7; R1 is H, alkyl or cation; m is 1-10; n is 1-5; and Y is H or D, where one or both Y is D. 6. The polyunsaturated substance for use according to any of claims 1-5, wherein a patient's cell or tissue maintains a sufficient concentration of the fatty acid or fatty acid ester to prevent autoxidation of the polyunsaturated fatty acid that occurs from natural or ester form. 7. The polyunsaturated substance for use according to any of claims 1-6, wherein the polyunsaturated substance is an omega-3 fatty acid, an omega-3 fatty acid ester, an omega-6 fatty acid or an ester of omega-6 fatty acid. 8. The polyunsaturated substance for use according to any of claims 1-7, wherein the polyunsaturated substance is selected from the group consisting of 11,11-D2-linolenic acid, 14,14-D2-linolenic acid, 11 ,11,14,14-D4-linolenic acid, 11,11-D2-linolenic acid, 11-D-linolenic acid, 14-D-linolenic acid, 11,14-D2-linolenic acid and 11-D-linolenic acid. 9. The polyunsaturated substance for use according to any of claims 1-3, wherein the polyunsaturated substance is a fatty acid ester selected from the group consisting of triglyceride, diglyceride, monoglyceride or alkyl ester. 10. The polyunsaturated substance for use according to any of claims 1-8, wherein the polyunsaturated substance is a fatty acid ester and the ester is an alkyl ester. 11. The polyunsaturated substance for use according to claim 10, wherein the alkyl ester is an ethyl ester. 12. The polyunsaturated substance for use according to any of claims 1-11, wherein the patient is classified as having steroid-induced cataract, steroid-induced myopathy, or Smith-Lemli-Opitz syndrome (SLOS), and the polyunsaturated substance is repeatedly administered to the patient beginning before, at the same time as, or after the administration of a steroid for the treatment of a disease or symptom. 13. The polyunsaturated substance for use according to any of claims 1-11, wherein the glaucoma is primary glaucoma, developmental glaucoma, secondary glaucoma or absolute glaucoma. 14. The polyunsaturated substance for use according to claim 13, wherein the glaucoma is primary angle-closure glaucoma, primary open-angle glaucoma, pigmentary glaucoma, exfoliation glaucoma, primary congenital glaucoma, infantile glaucoma, hereditary glaucoma, a glaucoma inflammatory glaucoma, phacogenic glaucoma, glaucoma secondary to intraocular hemorrhage, traumatic glaucoma, neovascular glaucoma, drug-induced glaucoma or glaucoma of various origins.